Red cell rinser

ABSTRACT

A red cell rinsing apparatus for preparing serological and like test specimens of sera including a liquid manifold having a plurality of ejection pipettes through which a plurality of microquantities of rinsing liquid are simultaneously ejected, and a carrier tray for supporting a plurality of red blood cell specimens arranged in rows. The tray is manually moved and located underneath the manifold successively to a plurality of predetermined portions in which the respective rows of specimens are automatically aligned with the ejection pipettes.

United States Patent [191 Gross RED CELL RINSER [75] Inventor: Ronald E. Gross, Springfield, Va.

[73] Assignee: The Cooke Engineering Company, Alexandria, Va.

22 Filed: on. 20, 1971 21 Appl.No.: 190,813

[52] US. Cl 23/259, 23/253 R, 23/258.5, 73/423 A, 141/244, 141/266, 141/283 [51] Int. CL... A61m l/03, B011 11/00, G01n 33/16 [58] Field of Search 23/258.5, 253 R; 141/244, 266; 73/423 A, 425.6

[56] References Cited UNITED STATES PATENTS 1,097,708 5/1914 Elson 23/259 2,772,531 12/1956 Brodsky 141/244 X 3,188,181 6/1965 Peterson et a1. 23/253 R X 3,351,432 11/1967 Van Dyck et a1... 23/258.5 3,399,040 8/1968 11g 23/258.5

[ 1 Dec.4, 1973 3,536,449 10/1970 Astle 23/253,R X 3,578,412 5/1971 Martin 23/259 3,622,279 ll/l971 Moran 231253 R X 3,650,306 3/1972 Lancaster 23/259 X 3,520,660 7/1970 Webb 195/127 Primary Examiner-Barry S. Richman Att0rneyStrauch, Nolan, Neale, Nies & Kurz [57] ABSTRACT A red cell rinsing apparatus for preparing serological and like test specimens of sera including a liquid manifold having a plurality of ejection pipettes through which a plurality of microquantities of rinsing liquid are simultaneously ejected, and a carrier tray for supporting a plurality of red blood cell specimens arranged in rows. The tray is manually moved and located underneath the manifold successively to a plurality of predetermined portions in which the respective rows of specimens are automatically aligned with the ejection pipettes.

13 Claims, 10 Drawing Figures PATENIEUnEc M915 I SHEET 10F 4 INVENTOR RONALD E. GROSS IWFNIEDDEC 4191s 3,776,699

sum 20? 4 FIG. 2

INVENTOR RONALD E. GROSS RNEY TMENIHIDEE 4 mm SHEET 3 BF 4 FIG. 5

INV ENTOR RONALD E. GROSS 'MWM PAIENIEUurn M975 3,776,699 v sum 4B? 4 INVENTOR RONALD E. GROSS JMWQ WW ATTORN YS tus and, more particularly, to a laboratory apparatus particularly useful in simultaneously rinsing aplurality of red blood cells to prepare serological and like test specimens of sera.

It is often necessary to rinse red blood cells to remove undesired biological matter from the exterior of the cells so that the cells arerendered suitable for test purposes.

In the past, red cell rinsing has been accomplished in closed system washers or rinsers in which macroquantities of red cells contained in bag-like containersare inundated and thoroughly mixed by means of stirring mechanism in substantial volumes of washing liquid or by manually positioning a single fluid ejection nozzle in succession over the individual wells of a microtitration plate in which each well contains a charge of red cells. The red cells, after thorough stirring with the washing liquid and after the stirrer is shut off, are allowed to settle out as a precipitate leaving an upper stratum of waste washing liquid and preservative and other removed matter which must be removed by draining it off to the line of demarcation between the lower stratum of red blood cells and the upper stratum of waste wash liquid. Typical examples of suchwashers or rinsers are illustrated in U.S. Letters Pat. No. 3,351,432 issued Nov. 7, 1967 to K. Van Dyck et al. and U.S. Pat. No. 3,399,040 issued Aug. 27, 1968 to J. C. Ilg. The rinsing operation in both prior art practices is time consuming and uniform rinsing is not assured.

SUMMARY OF THE INVENTION Accordingly, the primary object of this invention resides in the provision of a novel laboratory rinsing apparatus particularly useful for simultaneously rinsing a plurality of red blood cell samples distributed in microquantities in open wells of a plate such as a microtitration plate.

Another object resides in the provision of a relatively simple, inexpensive, manually operable laboratory rinsing apparatus in which each row of wells of a microtitration plate is automatically aligned underneath a plurality of wash fluid ejection pipettes so that the wells may be simultaneously filled with a rinsing solution.

Still another object resides in the provision of a novel laboratory rinsing apparatus as in the previous object, wherein the apparatus includes a manifold on which a plurality of pipettes are mounted and a movable carrier for supporting a microtitration plate which has a plurality of rows of red cell containing wells. The carrier is movable underneath the manifold and is automatically located and retained in a plurality of positions by an advance mechanism so that each row of wells is properly aligned with the pipettes to permitsimultaneous dispensing of a rinsing solution in all of the cells in each row.

Other objects and advantages of the invention will become apparent from reading the following descrip- FIG. 2-is a top plan view of the rinser shown in FIG. 1; f

FIG. 3 is a side elevation view of the rinser shown in FIG. 1;

FIG. 4 is a fragmentary front elevation view of the .rinser taken generally along line 4-4 of FIG. 3, with the syringe and rinsing liquid reservoir removed;

FIG; 5 is a bottom plan view of the base member of the rinser taken generally along line'5-5 of FIG. 3;

FIG. 6 is a fragmentary bottom plan view of the carrier tray on which the microtitration plateis supported, illustrating the manner in which the advancing and stop mechanism is connected to the carrier;

FIG. 7 is a fragmentary sectional view of the advancing and stop mechanism taken generally along line 77 of FIG. 6;

FIG. '8 is an enlarged perspective view of the manifold mounting bracket;

tion of a preferred embodiment, with reference to the drawings in which like numerals indicatelike elements. BRIEF DESCRIPTION OF THEDRAWINGS FIG. I is a general perspective view of the red cell rinser of the invention;

FIG. 9 is a perspective view of the locator bracket which fastens the fluid ejection manifold to the mounting bracket shown in FIG. 8; and

. FIG. 10 is a fragmentary sectional view of the fluid ejection manifold taken along line l010 of FIG. 2 and illustrating theinternal fluid passages of the manifold.

. DETAILED DESCRIPTION OF THE INVENTION wardly from base plate 24. A number of suction cups '34 are fixed to the bottom of base 24 to hold the chassis in place on a table or the like.

A pair of guide rails 36 and 38 are mounted on plate 24 along side flanges 26 and 28, respectively, and a carrier tray 40 is slidably supported along its outer edges 42 and 44 on rails 36 and 38, with the tray having downwardly depending front and rear flanges 46 and 48. A locator plate 50 is mounted along each of the tray edges 42 and 44, and each of plates 50 has a pair of projecting pins 52 which function to accurately position a microtitration plate on tray 40. The microtitration plate may be of any conventional type having a plurality of wells, but it is preferably of the type shown in U. S. Pat. No. 3,356,462 wherein there are eight rows of wells, with twelve wells in each row, thus providing a total of 96 wells in the plate.

Carrier tray 40 is sequentially advanced along rails 36 and 38 by an advance mechanism including a shaft '54 which slidably extends through flanges 30 and 32 of chassis 24 and flanges 46 and 48 of tray 40, with a 'nylon grommet 56 being fixed to each of the flanges and slidably receiving the shaft 54. A pushbutton knob 58 is fixed on the front end of shaft 54 and a spring 60 acts between flanges 30 and knob 58 to bias shaft 54 toward the front end to a normal home position. A

pair of set screw collars 62 are fixed on shaft 54 to establish the forwardmost home position of shaft 54 and to retain the shaft on chassis 22. Proper positioning of these set screw collars 62, particularly the one adjacent knob 58, axially along shaft 54 toward knob 58'is effected to establish the thrust stroke of the advance mechanism from the normal home position to a length slightly greater than the spacing between adjacent rows of wells. This will assure proper stepped advancement of carrier tray 40 from one preselected rinsing position to a point slightly beyond the next position so the biasing spring will be effective during the initial portion of the return stroke of the advance mechanism to firmly engage the stop mechanism, to be presently described, when the next row of wells is aligned below the ejector tips hereinafter described.

Shaft 54 is operatively connected to tray 40 to advance the tray when knob 58 is pushed inwardly. As best shown in FIGS. 1, 3, 4, 7 and 8, the connection is provided by an L-shaped spring member 64 having a horizontal leg 65 fixed to the underside of tray 40 by screws 66 which pass through the horizontal leg and thread into a spring support block 68 to fasten the block and spring member 64 against tray 40. The shaft 54 passes through the spacing between the vertical leg 70 of spring 64 and the opposing side of block 68, with leg 70 frictionally engaging shaft 54 to cause tray 40 to move rearwardly with the shaft when pushbutton 58 is depressed.

A stop lever or bar 72 is pivotally connected to the other side of block 68 via screw 74. The bar extends upwardly through a slot 75 in tray flange 46 and includes an upturned knurled handle section 76 and a lower projecting tooth 78 (FIG. 3) which is adapted to engage in a selected one of a plurality of detents or ridge abutments 80 provided in the base 24 of chassis 22. The spacing between ridges 80 corresponds to the spacing between the rows of wells in the microtitration plate which will be positioned on tray 40. When knob 58 is depressed, shaft 54 is moved rearwardly to advance tray 40 rearwardly (to the left in FIGS. 2 and 3) to the next position because of the friction engagement with spring leg 70. As this occurs, tooth 78 rides along base 24 and, when knob 58 is released, shaft 54 is returned to its forwardmost position by spring 60. However, tooth 78 will engage in one of the detents 80 to prevent return of tray 40 with shaft 54 which, as it returns, will slide relative to spring leg 70.

A wash fluid ejection assembly 82 is mounted on chassis 22 above tray 40 to simultaneously inject a predetermined 0.025 milliliter micro volume of rinsing liquid into all of the wells of a single row of wells in the microtitration plate. The assembly includes a right angle bracket 84 (FIG. 8) having a lower horizontal leg 86 which has a pair of laterally spaced openings 88. Leg 86 is connected to the bottom of base 24 via a pair of thumb screws 90, the shanks of which extend through openings 88, and aligned openings in base 24, and thread into tapped openings in a locator plate 92 positioned on the top of base 24. The openings 88 in bracket leg 86 are oversized with respect to the shanks of screws 90 so that bracket 86 is adjustable to permit proper lateral and longitudinal alignment of assembly 82 relative to detents 80 which, together with tooth 78, define the respective dispensing positions of carrier tray 40.

The vertical leg 94 of bracket 84 extends upwardly adjacent chassis flange 28 and supports at its uppermost end via block 96 an automatic rinse liquid pipette 98, which may be any known commercially available automatic pipetter such as a Cornwall syringe P5003 5. Syringe 98 is conventionally designed to draw liquid from a supply container 100 via tubing 102 connected to syringe 98 through a valve fitting 103 having an automatic one-way valve mechanism for alternately opening and closing the inlet and outlet,

branches in accord with the direction of movement of plunger 104. It delivers the rinse liquid via tubing 106 to a clear plastic dispensing manifold 108 (FIG. 10) upon the downstroke of plunger 104. The manifold 108 is supported from bracket leg 94 via a mounting bracket (FIG. 9) which has a vertical leg 112 provided with an integral threaded pem nut 114, with leg 112 being fastened to bracket leg 94 at a predetermined height above tray 40 by a screw 116 which extends through thefront one of slots 118 in leg 94. The transverse leg 120 of mounting bracket 110 also is provided with a pair of integral threaded pem nuts 122, and a pair of screws 124 extend through openings 126 in manifold 108 and thread into nuts 122 to fasten manifold 108 on bracket 110.

As shown in FIG. 10, manifold 108 includes a longitudinal passageway 128 which is intersected by a plurality of equally spaced vertical passageways 130. In the preferred embodiment illustrated, there are twelve vertical passageways 130 corresponding to the 12 wells normally provided in each row of wells in the conventional microtitration plates of the type shown in U. S. Pat. No. 3,356,462.

A disposable plastic pipette tip 132, designed to deliver a 0.025 charge of rinse liquid, is mounted in each passageway 130 to eject the liquid in twelve uniform micro streams that will successively fill each of the 12 well rows of a microtitration plate upon properly controlled step-by-step depression of plunger 104.

Manifold 108 also includes a small air bleed passage 134 which is intersected by a slide valve 136 that extends through manifold 108 from front to back, with a nut 138 threaded on the back end of the valve. The valve has a reduced center section 140, which when aligned with vent 134 bleeds air from passageway 128 as plunger 104 is activated to initially fill the manifold with liquid. When the manifold is filled, valve 136 is turned to block vent 134 and the manifold is ready for ejecting the liquid into the wells of the microtitration plate.

A typical red cell rinsing operation utilizing the present invention will now be described.

Manifold 108 is filled by actuating the pipette plunger 104 several times with valve 136 open to bleed any entrapped air from the manifold via vent 134. When the manifold is filled, valve 136 is closed readying rinser 20 for use. A microtitration plate, such as that shown in Letters Patent No. 3,356,462, with a previously deposited micro charge of red blood cells in each of its 96 wells, is placed on carrier tray 40 which is then advanced into position to bring the first row of twelve wells into position beneath the row of 12 pipette tips 132, preferably constructed in accord with the teachings of United States Letters Patent No. 3,276,639 issued Oct. 4, 1966 to Jesse F. Lancaster. Plunger 104 is then pressed downwardly in stepped sequence under operator control to simultaneously eject, under positive pressure, a stream of sufficient quantity of rinse liquid (about 0.025 milliliters from each tip 132) into the red cell mass in each plate well to forcefully disperse the rinse liquid into the red cell mass. The plate is advanced by actuation of pushbutton or knob 58 to its next position successively and repeated stepby-step until the red cells in the wells of the eight rows have been dispersed with rinse liquid.

The microtitration plate is then removed from carrier tray 40, a conventional cover tape is placed over the top surface of the microtitration plate to seal off each well from its neighboring wells, and the dispersion of red cells and rinse liquid in the 96 wells is conventionally simultaneously centrifugedbyplacing the taped plate in a centrifuge operating at about 1,000 RPM. This centrifuging operation thoroughly intermixes the red cells and rinse water in each well removing the cell contaminants and wetting the cells themselves with the protective rinse water.

After centrifuging, the red cells settle out from the rinse liquid into a precipitate in the form of a compacted mass of button shape in the bottom of each well. The cover tape is then removed and the excess rinse or waste liquid containing the undesirable contaminants is removed preferably by tilting the plate to an angle so that the microquantities of liquid in each cup can be poured off. Since the red cell masses at this stage are in the form of a silt-like wetted mass, they adhere to the bottom wall areas of the plate wells in proper position for transferring the plate containing the 96 rinsed red cell charges to further rinsing operations and finally the desired test procedure to be performed when the cells are sufficiently clean. Usually, three rinsings is sufficient to provide suitable test specimens.

Proper alignment of the successive rows of wells beneath pipette tips 132 is assured during the rinse liquid ejection procedure by the engagement and nesting of ratchet tooth 78 in the next successive detent 80 as the successive detents 80 are spaced a distance equal to the spacing between the successive rows of wells in the microtitration plate.

From the above description, it is apparent that the invention provides a single, inexpensive rinsing apparatus which is easily manipulated by a laboratory operation to quickly and accurately supply optimum microquantities of liquid into a predetermined test charge of red blood cell specimens deposited in a microtitration plate for ultimate test procedures effecting a substantial time saving and a saving of rinse solution and the test specimen supplies by reason of the microquantities employed.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiment is therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

What is claimed and desired to be secured by Letters Patent is:

1. Apparatus for preparing serological and like test specimens comprising a support base, carrier means movably mounted on said base, said carrier means being adapted to support a plurality of specimen receptacles arranged in a plurality of side-by-side rows, a fluid dispensing assembly mounted on said base and including a fluid dispensing manifold fixed above said carrier means and a plurality of dispensing elements connected to said manifold in transverse alignment for simultaneously dispensing-a quantity of fluid into each of said plurality of transversely aligned specimen receptacles, and advance means connected to said carrier means for advancing said carrier means relative to said base beneath said manifold sequentially through a plurality of predetermined positions in which the specimen porting said manifold from said base, said mounting bracket being adjustably connected to said base so that said manifold may be preferably adjusted relative to said locator means to ensure that said dispensing elements are preferably aligned with each row of specimens as said carrier means is moved to each of said predetermined positions.

4. Apparatus as defined in claim 3, said mounting bracket including a vertical leg to which said manifold is connected and a horizontal leg having at least one opening through which the shank of a fastening means extends to connect said horizontal leg to said base, said opening being oversized with respect to said shank to permit adjustment of said bracket and said manifold on said base.

5. Apparatus as defined in claim 4, said dispensing assembly comprising a locating bracket fixed to said manifold and adjustably connected to said vertical leg of said mounting bracket to adjust the height of said manifold above said carrier means.

6. Apparatus as defined in claim 3, said fluid dispensing assembly comprising a fluid source, a manually actuated pipettor mounted on said mounting bracket,

'conduit means connecting said pipettor to said source and said manifold to fill said manifold, and said manifold including a bleed valve assembly for venting air from said manifold as said manifold is being-filled.

7. Apparatus as defined in claim 6 wherein said manually actuated pipettor isof the reciprocating plunger type adapted to withdraw fluid from said source upon movement of said plunger in one direction and to dispense fluid into said manifold upon movement of said plunger in. the other direction and is operative, when said manifold is filled with fluid and said bleed valve is closed, to eject measured quantities of fluid from said manifold through said plurality of dispensing elements upon controlled movement of said plunger by the operator.

8. Apparatus for preparing serological and like test specimens comprising a support base; carrier means mounted on said support base for movement between opposite ends of said base, said carrier means being adapted to support a plurality of specimen receptacles arranged in a plurality of side-by-side rows; a fluid dispensing assembly mounted on said base and including a fluid dispensing manifold fixed above said carrier means and a plurality of dispensing elements connected to said manifold for dispensing a quantity of fluidinto each of said plurality of aligned specimen receptacles; advance means comprising manually operated actuator drive means to advance said carrier means relative to said base through a plurality of predetermined positions in which the specimen receptacles of adjacent rows are successively aligned with said dispensing elements andlocator means associated with said carrier means to automatically locate said carrier means sequentially in said predetermined positions, said actuator drive means including a shaft extending between said opposite ends of said base, spring means for biasing said shaft in one direction to a home position and means for drive connecting said shaft to said carrier means so that, when said shaft is manually moved in the other direction, said carrier means is advanced therewith from one preselected position to another preselected position, and said locator means comprising stop means associated with said carrier means and said base for positioning said carrier means in a next predetermined position when said shaft is released to return to said home position by said spring means.

9. Rinsing apparatus as defined in claim 8, said drive connecting means comprising friction drive means coupling said shaft to said carrier means.

10. Apparatus as defined in claim 9, said friction drive means being formed by an L-shaped spring element one leg of which is connected to said carrier means and the other leg of which frictionally engagescomprising a stop member pivotally connected to said carrier means and a plurality of longitudinally spaced locating abutments on said base disposed for ratchetting cooperation with said abutments during manual movementof said shaft and abutting cooperation to establish said predetermined positions of said carrier means along said base upon release of said shaft so that each row of specimen is accurately aligned with said dispensing elements.

12. Apparatus as defined in claim 11, said stop member having one end pivotally connected to said carrier means and a depending lug spaced from said one end for engaging a respective lug of one of said abutments to prevent return movement of said carrier means with said actuating shaft when said shaft is released.

13. Apparatus as defined in claim 12, wherein said stop member extends beyond said lug to provide a hand hold portion that may be grasped to pivot said stop member in a direction to raise said lug clear of said abutment and thereby permit returnof said carrier means toward said one end. 

2. The apparatus as defined in claim 1, said advance means comprising manually operated actuator drive means connected to said carrier means to advance said carrier means from one position to another, and locator means associated with said carrier means to automatically locate said carrier means in a selected one of said predetermined positions.
 3. Apparatus as defined in claim 1, said fluid dispensing assembly comprising a mounting bracket for supporting said manifold from said base, said mounting bracket being adjustably connected to said base so that said manifold may be preferably adjusted relative to said locator means to ensure that said dispensing elements are preferably aligned with each row of specimens as said carrier means is moved to each of said predetermined positions.
 4. Apparatus as defined in claim 3, said mounting bracket including a vertical leg to which said manifold is connected and a horizontal leg having at least one opening through which the shank of a fastening means extends to connect said horizontal leg to said base, said opening being oversized with respect to said shank to permit adjustment of said bracket and said manifold on said base.
 5. Apparatus as defined in claim 4, said dispensing assembly comprising a locating bracket fixed to said manifold and adjustably connected to said vertical leg of said mounting bracket to adjust the height of said manifold above said carrier means.
 6. Apparatus as defineD in claim 3, said fluid dispensing assembly comprising a fluid source, a manually actuated pipettor mounted on said mounting bracket, conduit means connecting said pipettor to said source and said manifold to fill said manifold, and said manifold including a bleed valve assembly for venting air from said manifold as said manifold is being filled.
 7. Apparatus as defined in claim 6 wherein said manually actuated pipettor is of the reciprocating plunger type adapted to withdraw fluid from said source upon movement of said plunger in one direction and to dispense fluid into said manifold upon movement of said plunger in the other direction and is operative, when said manifold is filled with fluid and said bleed valve is closed, to eject measured quantities of fluid from said manifold through said plurality of dispensing elements upon controlled movement of said plunger by the operator.
 8. Apparatus for preparing serological and like test specimens comprising a support base; carrier means mounted on said support base for movement between opposite ends of said base, said carrier means being adapted to support a plurality of specimen receptacles arranged in a plurality of side-by-side rows; a fluid dispensing assembly mounted on said base and including a fluid dispensing manifold fixed above said carrier means and a plurality of dispensing elements connected to said manifold for dispensing a quantity of fluid into each of said plurality of aligned specimen receptacles; advance means comprising manually operated actuator drive means to advance said carrier means relative to said base through a plurality of predetermined positions in which the specimen receptacles of adjacent rows are successively aligned with said dispensing elements and locator means associated with said carrier means to automatically locate said carrier means sequentially in said predetermined positions, said actuator drive means including a shaft extending between said opposite ends of said base, spring means for biasing said shaft in one direction to a home position and means for drive connecting said shaft to said carrier means so that, when said shaft is manually moved in the other direction, said carrier means is advanced therewith from one preselected position to another preselected position, and said locator means comprising stop means associated with said carrier means and said base for positioning said carrier means in a next predetermined position when said shaft is released to return to said home position by said spring means.
 9. Rinsing apparatus as defined in claim 8, said drive connecting means comprising friction drive means coupling said shaft to said carrier means.
 10. Apparatus as defined in claim 9, said friction drive means being formed by an L-shaped spring element one leg of which is connected to said carrier means and the other leg of which frictionally engages said shaft.
 11. Apparatus as defined in claim 8, said stop means comprising a stop member pivotally connected to said carrier means and a plurality of longitudinally spaced locating abutments on said base disposed for ratchetting cooperation with said abutments during manual movement of said shaft and abutting cooperation to establish said predetermined positions of said carrier means along said base upon release of said shaft so that each row of specimen is accurately aligned with said dispensing elements.
 12. Apparatus as defined in claim 11, said stop member having one end pivotally connected to said carrier means and a depending lug spaced from said one end for engaging a respective lug of one of said abutments to prevent return movement of said carrier means with said actuating shaft when said shaft is released.
 13. Apparatus as defined in claim 12, wherein said stop member extends beyond said lug to provide a hand hold portion that may be grasped to pivot said stop member in a direction to raise said lug clear of said abutment and thereby permit return of said carrier means toward said one end. 